One example of progress we recently made is in cryo-EM structure determination of beta-galactosidase, in complex with phenylethyl beta-D-thiogalactopyranoside (PETG), a potent inhibitor that blocks enzyme activity by replacing the oxygen in the O-glycosidic bond with a sulfur atom. Our map at a resolution of 2.2 Angstroms showed clear densities for several backbone carbonyl groups and several ordered water molecules in the structure. In the vast majority of instances, these water molecules are at locations also identified in the 1.7 Angstrom crystal structure of beta-galactosidase (PDB 1DP0), providing independent validation of their assignment. This work was published in Science in 2015. In another important advance, our collaboration led to breaking of both resolution and size barriers in the cryo-EM field. Using the dehydrogenase enzymes as a paradigm for small metabolic protein complexes, we showed that the structures of enzyme complexes as small as 93 KDa can be analyzed using cryo-EM at near-atomic resolution. We also showed that resolutions as high as 1.8 Angstrom can be obtained even with enzymes such as glutamate dehydrogenase, which have highly flexible outer regions. In a third example, we showed that complexes of the 145 kDa lactate dehydrogenase with an inhibitor can be determined at resolutions better than 3 Angstroms. These results, which provide new hope that cryo-EM methods can provide important mechanistic information for drug discovery without the need for crystallization, were published in the journal Cell in 2016. In more recent work, we are beginning to apply cryo-EM methods to another metabolic enzyme PKM2, that is also a cancer target. Key metabolic reprogramming events in cancer cells include the upregulation of glycolysis regardless of oxygen availability as well as a switch in expression from the M1 to the M2 isoform of pyruvate kinase. The H391Y mutation in PKM2 identified in cancer-prone Bloom Syndrome (BS) patients has been shown to reduce pyruvate kinase activity. By determining the near-atomic resolution structures of both wild-type and H391Y variants, we show that the mutation does not result in any significant perturbation of the tertiary or quaternary structure of the enzyme. Efforts are underway to map the subtle changes in the active site introduced by the mutation, as well as the effects of the mutation on binding substrates, activators and inhibitors.